The work proposed in this application is designed to provide insight into the molecular mechanisms that underlie a fundamental regulatory pathway in the heart: control of cardiac electrical activity by the sympathetic nervous system (SNS). To ensure adequate diastolic filling time between heartbeats during exercise and stress when SNS activity is increased, the duration of depolarization of the ventricular chambers, the QT interval of the electrocardiogram must be shortened. This occurs in large part through an increase in repolarization reserve of the heart by a protein kinase A (PKA) mediated regulation of the slowly activating IKS potassium (K+) channel, a process that requires assembly of a multi protein signaling complex coordinated by the A-Kinase anchoring protein Yotiao. That this K+ channel and its regulation are critical to human cardiac electrophysiology is evident from the range of heritable arrhythmias linked to mutations in genes coding for its principle subunits or for channel-associated proteins that coordinate its regulation. The long-term objective of this project is to identify additional signaling molecules that comprise the IKS multi protein complex, to unravel the fundamental processes by which these molecules control the IKS channel, and to relate them to our understanding of the basis and treatment of human disease. There are four aims of the proposed work. Aim 1 is to identify additional signaling molecules and their binding motifs in the IKS signaling complex which we postulate will serve as templates for discovery of, as yet unidentified, inherited Yotiao mutations that underlie multiple heritable arrhythmia syndromes. Aim 2 is to test the hypothesis that the SNS-mediated increase in repolarization reserve in the heart is due in part to an increase in the number of functional IKS channels regulated by a phosphorylation-sensitive trafficking pathway. Aim 3 is to test the hypothesis that SNS regulation of the IKS channel, physiologically essential in healthy individuals, can be a critical contributor to arrhythmia susceptibility in inherited atrial arrhythmia syndromes. Aim 4 is to test the hypothesis that IKS channels are expressed and regulated in cardiac myocytes (CMs) differentiated from human embryonic stem cells (hESCs) and that these cells can serve as a novel model system for investigating the expression and regulation of this and other critical channels in a human cardiac cellular environment. We propose that characterization of these channels in hESC-derived CMs will serve as an important baseline for future studies of these channels in inducible pluripotent stem cells (iPSCs) derived from patients suffering from heritable arrhythmia syndromes. The proposed project will combine biochemistry, imaging, and single cell electrophysiology in human cardiac cells to provide insight into the mechanisms causing congenital arrhythmias and, consequently, more specific and effective strategies to treat them.